Functional Characterization of Integrin α6β4 Adhesion Interactions Using Soluble Integrin Constructs Reveals the Involvement of Different Functional Domains in the β4 Subunit

Abstract

Integrin a6b4Ámediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how a6b4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing a6b4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact a6b4. Using these reagents in combination with anti-b4 antibodies, the authors identified two distinct functional epitopes on the b4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in a6b4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating a6b4 signaling biology.

Integrin-Blocking Antibodies Delay Keratinocyte Re-Epithelialization in a Human Three-Dimensional Wound Healing Model

“…This sub-basal split seen under the stratum basale was similar to the pattern previously reported from the skin of patients with junctional epidermolysis bullosa, in which mutation of the β4 subunit resulted in separation of the epithelium at the basement membrane interface [25], [26]. Interestingly, the non-adhesion-blocking antibody, ASC-3, demonstrated anti-tumor effects in vitro in our prior studies [15], supporting the conclusion that the tumorigenic properties of β4 can be targeted independently of the adhesive properties to laminins.…”

“…Therefore, we chose the well described HSE tissue model, which recapitulates human skin and wound healing, to test antibodies directed at β4. Our previous results [15] demonstrated that the adhesion-blocking antibody ASC-8, had no effect on anchorage-independent growth of tumor cells, but a non-adhesion blocking antibody, ASC-3, did inhibit this β4 mediated function. We have extended these studies to show that ASC-8 disrupts the attachment of basal keratinocytes at the basement membrane interface and has a profound effect on wound closure by blocking migration of keratinocytes in the epithelial tongue of wounded HSEs.…”

“…We previously demonstrated that an antibody (ASC-3) directed to β4 could prevent anchorage-independent growth of breast tumor cells in preclinical assays [15]. The activity and epitope of this antibody was distinguishable from an adhesion blocking antibody (ASC-8) that disrupts β4 interactions with laminin-5, but does not affect growth in soft agar.…”

Antibodies directed to α6β4 highlight the adhesive and signaling functions of the integrin in breast cancer cell lines

“…Recombinant α6β4 CHO expressing cell lines and soluble α6β4 Fc proteins were generated as described previously. 27 AIIB2 (anti-β1) antibody was purified by standard protein G affinity chromatography from hybridoma cells cultured following the supplier's protocol (Developmental Studies Hybridoma Bank at the University of Iowa, IA). Anti-EGFR antibody (225) was purchased from EMD Biosciences (San Diego, CA).…”

“…Previously we generated recombinant soluble forms of α6β4 ectodomains and confirmed these proteins retained the properties of the intact integrin. 27 We next generated a panel of Fabs recognizing these proteins by panning a human phage display library. Two Fabs encoding distinct variable domain regions were converted to full length antibodies and characterized by flow cytometry.…”